Dipeptides are used in the field of pharmaceutical materials and functional foods and various fields. For example, L-alanyl-L-glutamine is used as a component of serum-free media, and is used for infusion components since it has greater stability and higher solubility than L-glutamine.
Chemical synthesis methods, which have been conventionally known as methods of producing dipeptides, are not necessarily simple. Known examples of such methods include a method that uses N-benzyloxycarbonylalanine (hereinafter, “Z-alanine”) and protected L-glutamine (see Bull. Chem. Soc. Jpn., 34, 739 (1961), Bull. Chem. Soc. Jpn., 35, 1966 (1962)), a method that uses Z-alanine and protected L-glutamate-γ-methyl ester (see Bull. Chem. Soc. Jpn., 37, 200 (1964)), a method that uses a Z-alanine ester and unprotected glutamic acid (see Japanese Patent Application Laid-Open Publication No. H1-96194), and a method that uses a 2-substituted-propionyl halide as raw material and synthesizes an N-(2-substituted)-propionyl glutamine derivative as an intermediate (see Japanese Patent Application Laid-Open Publication No. H6-234715).
However, in all of these methods, the introduction and elimination of a protecting group or the synthesis of an intermediate is required, so that these production methods have not been sufficiently satisfactory in view of their industrial advantages. Known examples of typical dipeptide production methods using enzymes include a condensation reaction using an N-protected, C-unprotected carboxy component and an N-unprotected, C-protected amine component (Reaction 1), and a substitution reaction using an N-protected, C-protected carboxy component and an N-unprotected, C-protected amine component (Reaction 2). An example of Reaction 1 is a production method of Z-aspartylphenylalanine methyl ester from Z-aspartic acid and phenylalanine methyl ester (see Japanese Patent Application Laid-Open Publication No. S53-92729), while an example of Reaction 2 is a production method of acetylphenylalanylleucine amide from acetylphenylalanine ethyl ester and leucine amide (see Biochemical J., 163, 531 (1977)). There are extremely few examples of research reports that describe methods using N-unprotected, C-protected carboxy components. An example of a substitution reaction using an N-unprotected, C-protected carboxy component and an N-unprotected, C-protected amine component (Reaction 3) is described in Patent WO 90/01555, and example of such a reaction is a production method of arginyl leucine amide from arginine ethyl ester and leucine amide. An example of a substitution reaction using an N-unprotected, C-protected carboxy component and an N-unprotected, C-unprotected amine component (Reaction 4) is described in Patent EP 278787A, and example of such a reaction is a production method of tyrosyl alanine from tyrosine ethyl ester and alanine. Among the methods those that are able to serve as the least expensive production methods are naturally those that fall in the range of Reaction 4 involving the fewest number of protecting groups.
However, the enzyme used in the example of the prior art of the Reaction 4 (see Patent EP 278787A) is a comparatively expensive carboxypeptidase preparation derived from molds and plants, and the dipeptides that were produced contained amino acids that are comparatively highly hydrophobic. For the Reaction 4, there is no known method that uses an enzyme of bacterial or yeast origin, and there has been known no method.for producing highly hydrophilic alanylglutamine or alanylasparagine. Under such circumstances, there has been a need for the development of an inexpensive industrial method for the production of such peptides.
On the other hand, proline iminopeptidase is an enzyme that catalyzes a reaction that cleaves an N-terminal proline from a peptide having proline on its N-terminal, and this enzyme is known to exist in numerous species of organisms. For example, it is known to exist in higher animals such as guinea pigs (brain) (see J. Biol. Chem., 258, 6147-6154 (1983)), rats (brain and kidneys) (see Eur. J. Biochem., 190, 509-515 (1990)), higher plants such as apricot seeds (see J. Biochem., 92, 413-421 (1982)), oral cavity spirochetes such as Trichoderma denticola (see Infect. Immun., 64, 702-708 (1996), filamentous fungi such as Penicillium species (see Japanese Patent Application Laid-Open Publication No. H1-215288), Basidiomycetes such as shiitake mushrooms (see Japanese Patent Application Laid-Open Publication No. S58-36387), Actinomycetessuch as Streptomyces plicatus (see Biochem. Biophys. Res. Commun., 184, 1250-1255 (1992), and bacteria such as Corynebacterium variabilis (see J. Appl. Microbiol., 90, 449-456 (2001)).
In addition, concerning proline iminopeptidase gene, there have been reported cloning and base sequences of genes of Arthrobacter nicotiana (see FEMS Microbiol. Lett., 78, 191-197 (1999)), Escherichia coli (see Japanese Patent Application Laid-Open Publication No. H2-113887), Flavobacterium meningosepticum (see Arch. Biochem. Biophys., 336, 35-41 (1996)), Hafnia alvei (see J. Biochem., 119, 468-474 (1996)), Lactobacillus delbrueckii (see Microbiology, 140, 527-535 (1994)), Bacillus coagulans source (see J. Bacteriol., 174, 7919-1925 (1994)), Aeromonas sobria source (see J. Biochem., 116, 818-825 (1994)), Xanthomonas campestris (see Japanese Patent Application Laid-Open Publication No. H9-121860), Neisseria gonorrhoeae (see Mol. Microbiol., 9, 1203-1211 (1993), Propionibacterium freundenreichii (see Appl. Environ. Micorbiol., 64, 4736-4742 (1998)), Serratia marcescens (see J. Biochem., 122, 601-605 (1997)) and Thermoplasma acidophilum (see FEBS Lett., 398, 101-105 (1996)).
In addition, base sequences predicted to encode proline iminopeptidase have recently been reported in numerous species of organisms as a result of analyses on the whole genomes of microbes. For example, the whole genome base sequence of Pseudomonas aeruginosa has been reported (see Nature, 406, 959 (2000)), and a base sequence was found therein that is predicted to encode proline iminopeptidase.
On the other hand, it has been found that a proline-containing dipeptide is formed when an ester of L-proline or DL-proline and an alpha-amino acid are allowed to react using proline iminopeptidase (see Japanese Patent Application Laid-Open Publication No. H3-13391). However, although proline iminopeptidase is an enzyme that catalyzes a reaction that cleaves the N-terminal proline from a peptide having proline on its N-terminal, and a prolyl amino acid would be naturally considered to be produced from proline ester and amino acid, the synthesis of peptide from an amino acid and amino acid ester other than proline using proline iminopeptidase has been completely unknown. Of course, the synthesis of L-alanyl-L-glutamine from L-alanine ethyl ester hydrochloride and L-glutamine has been also previously unknown. In addition, although the partial base sequence of proline iminopeptidase of Pseudomonas putida strain ATCC 12633 was disclosed (AF032970), there has been no study conducted whatsoever on its activity, including its detection.